Transmission of malaria from one human to another solely depends upon the successful development of the parasite in the vector mosquitoes. If a mosquito does not allow complete sporogonic development, the parasite dies, and the transmission of malaria is blocked. Deliberate interference of the malaria parasite development in vector mosquitoes is a proposed strategy to block the spread of malaria. For designing such strategies, however, detailed understanding of the parasite developmental biology in the vector mosquitoes is essential. The purpose of this project is to understand the biology of the malaria parasite in the vector mosquito to help development of strategies against malaria transmission. The development of malaria parasites in the mosquito is complex and poorly understood. After being ingested by a susceptible mosquito, the parasite undergoes elaborate developmental changes. These include, sequential morphological changes, expression of stage-specific parasite genes and interaction of the parasite with various tissues and molecules in the mosquitoes. The development is, therefore, a huge challenge to the ingested parasite, and most parasites fail to develop successfully even in a most successful vector. The goal of this project is to identify crucial parasite and mosquito molecules that determine the successful development of malaria parasite. In the mosquito, most of the developmental changes occur to the parasites while they are associated with the midgut. Our current interest, therefore, is focused on the midgut stages of the parasite. We are studying why most of the ingested parasites fail to complete the development, how the parasite recognizes midgut epithelium as a target tissue for invasion, how the parasite invades the epithelial cells, how the developmental switch from ookinete to oocysts occurs and how the parasite survives and develops as a mature oocyst in the hemolymph of the vector. This project will lead to acquiring molecular information that can be exploited to block malaria transmission. Following is the summary of specific work areas: 1. We have identified a factor in the mosquitoes that fully blocks the transformation of the zygote to ookinetes. The factor does not kill the parasite, but blocks elongation of the ookinete. It is about 502 Dalton in size and not an oligopeptide. Mass spectrophotometry and NMR did not show similarity with any known compounds. This is a first putative antimalarial factor purified from mosquitoes. The chemical structure of the compound is being determined. 2. Scanning electron microscopy revealed a complex surface structure of the mosquito midgut lumen. Based on the structural differences, midgut cells can be classified into a number of cell types. We have identified several changes that occur in response to the blood meal. This information is important in determining the cell type that malaria parasite interacts with before and during invasion. 3. We have shown that ookinete binding to the epithelial cell surface is different than binding to the lumen-associated network-like structure. The network-like structure appears to be related to the peritrophic matrix, and the parasite binds with higher affinity with this structure. These results suggested that ookinete invasion of the midgut occurs after a sequential adhesion to a variety of structures in the gut lumen. 4. Previously, we found a sialic acid-like surface carbohydrate moiety as a potential molecule for ookinete recognition of the midgut epithelium. Exhaustive search using various chromatographic and biochemical assays did not show the presence of sialic acid in the mosquito. This suggests that the recognition receptor is not a sialic acid-containing molecule, but shares some biochemical properties with sialic acids. 5. We have previously shown that vesicular ATPase is overexpressed in midgut cells that malaria parasites prefer to invade. We have now cloned the B-subunit of the enzyme from Aedes aegypti and made antiserum to the recombinant protein. The antiserum also recognizes Anopheles gambiae V-ATPase. This has allowed us to further study the V-ATPase overexpressing cells in both Aedes and Anopheles mosquitoes. 6. We have found that spatial distribution of the v-ATPase overexpressing cells is similar in Aedes and Anopheles mosquitoes. The distribution pattern of the cells matches the typical distribution pattern of the Plasmodium oocysts, which is usually found close to the posterior end of the posterior midgut. This suggested that the oocyst distribution pattern is due to the prevalence of the special types of cells in this region of the gut. Previously, it was thought that the effect of gravity on the vertically aligned mosquitoes determines the pattern. 7. We have developed in vivo techniques to study the invasion efficiency of Plasmodium parasites in different mosquitoes and preliminary data suggest that invasion in susceptible mosquitoes is more efficient. This method has allowed us to determine the precise time of maximum ookinete invasion of midgut epithelium after the ingestion of the parasite. 8. We have prepared 92 monoclonal antibodies with midgut epithelial plasma membranes as antigens. Of these, 29 have been cloned and are being used to study the location of the antigens and their role in Plasmodium development. Two of these monoclonal antibodies recognize antigens specifically located on the brush border of the gut lumen and on Peritrophic matrix. Other monoclonal antibodies labeled sites including basal lamina and intracellular organelles. The antigens that are recognized by these monoclonal antibodies are being further characterized for location of expression and their relationship with Plasmodium development.